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US 10,851,362 B2Granted
Intein-modified proteases, their production and industrial applications
AssigneeAgrivida, Inc.
InventorsR. Michael Raab, Binzhang Shen, Gabor Lazar, Humberto De La Vega
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Abstract
Methods for producing intein-modified proteases are provided. Expression cassettes and vectors for using to genetically engineer hosts are described. Hosts genetically engineered to express one or more intein-modified proteases using expression cassettes and vectors of the invention are also provided. Methods to produce a protease and regulate its activity are described.
Claims13
An animal feed comprising a first portion and a second portion of an intein-modified protease,
- wherein the first portion of the intein-modified protease comprises an N-extein of a target protease and an N-intein of an intein, and a carboxy terminus of the N-extein is fused to an amino terminus of the N-intein, the second portion of the intein-modified protease comprises a C-intein of the intein and a C-extein of the target protease, and a carboxy terminus of the C-intein is fused to an amino terminus of the C-extein, wherein the N-intein includes a sequence with at least 90% identity to the reference sequence of SEQ ID NO:
- 537, and the C-intein includes a sequence with at least 90% identity to the reference sequence of SEQ ID NO:
- 538, and
- wherein the target protease comprises a sequence with at least 90% identity to the reference sequence of SEQ ID NO:
- 57, and has a serine at a position aligned to position 109, 267, or 291 of SEQ ID NO:
- 57, or a threonine at a position aligned to position 292 of SEQ ID NO:
- 57, and the C-intein is fused to an amino acid residue aligned to position 109, 243, 267, 291 or 292 of SEQ ID NO:
- 57 of the target protease, each of the first portion and the second portion is in a dry form, the first portion is separated from the second portion, and the intein is capable of effecting trans-splicing of the intein modified protease upon mixing of the first portion and the second portion to form a mixture and hydration of the mixture.
5 more elements
An expression cassette comprising:
- a polynucleotide encoding at least one of i) a first portion of an intein-modified protease that includes a N-extein of a target protease and an N-intein of an intein, and a carboxy terminus of the N-extein is fused to an amino terminus of the N-intein, and ii) a second portion of an intein-modified protease having a C-intein of the intein and a C-extein of the target protease, and a carboxy terminus of the C-intein is fused to an amino terminus of the C-extein,
- wherein a nucleotide sequence having at least 90% identity to the reference sequence of SEQ ID NO:
- 674 encodes the N-intein, and a nucleotide sequence having at least 90% identity to the reference sequence of SEQ ID NO:
- 675 encodes the C-intein,
- wherein the N-extein comprises a sequence with at least 90% identity to a first sequence that is one of positions 1-108, 1-242, 1-266, 1-290 or 1-291 of the reference sequence of SEQ ID NO:
- 57, and
- wherein the C-extein comprises a sequence with at least 90% identity to a second sequence that is one of positions 109-354, 243-354, 267-354, 291-354 or 292-354 of the reference sequence of SEQ ID NO:
- 57, and has a serine at a position aligned to position 109, 243, 267, or 291 of SEQ ID NO:
- 57 or a threonine at a position aligned to position 292 of SEQ ID NO:
- 57, and the C-intein is fused to an amino acid residue aligned to position 109, 243, 267, 291 or 292 of SEQ ID NO:
- 57.
8 more elements
Dependencies are inferred from the claim text.